Abstract / Summary 240 million Americans and 2 billion people worldwide are obese or overweight; this is a major health concern, associated with heart disease, cancer, and diabetes. Obesity has proven difficult to treat, but engineered microbes show promise. E. coli engineered to produce N-acyl phosphatidylethanolamines (NAPEs) resulted increased satiety, decreased food intake, decreased body fat, and decreased insulin resistance in obese mice. Unfortunately, lack of safety and containment measures in this organism precludes its clinical evaluation and use in humans. A robust biosafety solution to address control and containment concerns would unlock the therapeutic potential of this organism. The optimal solution may be synthetic auxotrophs: organisms engineered to require the presence of a complementing ligand (ligand). The principal investigator (Dr. Lopez) demonstrated a method for generating synthetic auxotrophs based on a Synthetic Ligand-Dependent Essential gene (SLiDE allele). SLiDE strains are an ideal biosafety solution because they ensure engineered organisms can be ?turned off? by removing the ligand. They also prevent organisms from escaping into the environment by requiring that organisms only grow where ligand is supplied. This will broadly enable the use of therapeutic organisms by providing a versatile, reliable biosafety technology. Incorporation of NAPE production into SLiDE biosafety strains will produce a therapeutic organism with intrinsic biosafety, but the ligand-dependence of the SLiDE organism must be altered to food-safe compounds for use in patients. This will be achieved by applying directed evolution to force the SLiDE strain to control its viability with food-safe ligands and ignore other compounds found in nature and the gut. The products of this Phase I grant, NAPE-producing SLiDE strains that require food-safe ligands for continued viability, would represent a critical step in developing an anti-obesity therapeutic organism safe for use in patients. The combination of anti-obesity efficacy with the unprecedented ability to prevent therapeutic organisms from escaping and turn off therapy on demand would be a valuable achievement for engineered probiotics. The most promising strains generated in this study will be ideal for further characterization in vivo in Phase II, with an eye towards eventual clinical trials.